Electroporation microarray for parallel transfer of small interfering RNA into mammalian cells.
This paper describes the fabrication of microarrays that enable the parallel electroporation of small interfering RNAs (siRNAs) into mammalian cells. To optimize the conditions of microarray preparation and electric pulsing, a self-assembled monolayer was formed on a gold electrode, and a cationic polymer was adsorbed by the entire surface of the monolayer. siRNA was then adsorbed by the cationically modified electrode through electrostatic interactions. Human embryonic kidney cells stably transformed with the expression construct of green fluorescent protein (GFP) were used to examine the electric pulse-triggered transfer of GFP-specific siRNA. A single electric pulse was applied to the cells cultured on the electrode at a field strength of 240 V cm(-1). The expression of GFP was significantly suppressed in a sequence-specific manner two days after pulsing. Microscopic observation and flow-cytometric analysis revealed that the expression of GFP was attenuated in the majority of cells in a loading-dependent manner. Moreover, the effect of siRNA could be temporally controlled by changing the culture periods before pulsing. When a micropatterned self-assembled monolayer was used as a platform for loading siRNA in an array format, gene silencing was spatially restricted to the regions where specific siRNA was loaded. From these results, we conclude that array-based electroporation provides an excellent means of individual transfer of siRNAs into mammalian cells for high-throughput gene function studies.
Fujimoto
Hiroyuki
H
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Kato
Koichi
K
Iwata
Hiroo
H
D002460:Cell Line; D004566:Electrodes; D018274:Electroporation / Q000379:methods* / Q000592:standards; D005434:Flow Cytometry; D020868:Gene Silencing; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D034741:RNA, Small Interfering / Q000008:administration & dosage / Q000493:pharmacokinetics*
D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins